Molecular Basic of Inheritance

CAMPBELL BIOLOGY IN FOCUS
URRY • CAIN • WASSERMAN • MINORSKY • REECE
13
The Molecular
Basis of Inheritance
Lecture Presentations by
Kathleen Fitzpatrick and
Nicole Tunbridge,
Simon Fraser University
© 2016 Pearson Education, Inc. SECOND EDITION

Overview: Life’s Operating Instructions
 In 1953, James Watson and Francis Crick

introduced an elegant double-helical model for the
structure of deoxyribonucleic acid, or DNA
 DNA, the substance of inheritance, is the most
celebrated molecule of our time
 Hereditary information is encoded in DNA and
reproduced in all cells of the body (DNA
replication)
© 2016 Pearson Education, Inc.

Figure 13.1

Figure 13.2

Concept 13.1: DNA is the genetic material
 Early in the 20th century, the identification of the

molecules of inheritance loomed as a major
challenge to biologists

The Search for the Genetic Material: Scientific
Inquiry
 When T. H. Morgan’s group showed that genes are
located on chromosomes, the two components of
chromosomes—DNA and protein—became
candidates for the genetic material
 The key factor in determining the genetic material
was choosing appropriate experimental organisms
 The role of DNA in heredity was worked out by
studying bacteria and the viruses that infect them

Evidence That DNA Can Transform Bacteria
 The discovery of the genetic role of DNA began with

research by Frederick Griffith in 1928
 Griffith worked with two strains of a bacterium, one
pathogenic and one harmless

 When he mixed heat-killed remains of the
pathogenic strain with living cells of the harmless
strain, some living cells became pathogenic
 He called this phenomenon transformation, now
defined as a change in genotype and phenotype
due to assimilation of foreign DNA

Figure 13.3
Experiment
Living S cells Living R cells Heat-killed S cells Mixture of heat-
(control) (control) (control) killed S cells and
living R cells
Results
Mouse dies Mouse healthy Mouse healthy Mouse dies
Living S cells

 Later work by Oswald Avery and others identified
the transforming substance as DNA
 Many biologists remained skeptical, mainly because
little was known about DNA and they thought
proteins were better candidates for the genetic
material

Evidence That Viral DNA Can Program Cells
 More evidence for DNA as the genetic material

came from studies of viruses that infect bacteria
 Such viruses, called bacteriophages (or phages),
are widely used in molecular genetics research
 A virus is DNA (or RNA) enclosed by a protective
protein coat
 Viruses must infect cells and take over the cells’
metabolic machinery in order to reproduce

Figure 13.4
Phage
head
DNA
Tail
sheath
Tail fiber
Genetic
material
100 nm
Bacterial
cell

 In 1952, Alfred Hershey and Martha Chase showed
that DNA is the genetic material of a phage known
as T2
 To determine this, they designed an experiment
showing that only the DNA of the T2 phage, and not
the protein, enters an E. coli cell during infection
 They concluded that the injected DNA of the phage
provides the genetic information

Figure 13.5
Experiment
Batch 1: Radioactive sulfur (35S) in phage protein
Labeled phages Agitation frees outside Centrifuged cells form
infect cells. phage parts from cells. a pellet. Free phages
and phage parts remain
in liquid.
Radioactive Radioactivity
protein (phage protein)
found in liquid
DNA
Centrifuge
Pellet
Batch 2: Radioactive phosphorus (32P) in phage DNA
Radioactive
DNA
Centrifuge
Radioactivity (phage
Pellet DNA) found in pellet
Figure 13.5-1
Experiment
Batch 1: Radioactive sulfur (35S) in phage protein
Labeled phages Agitation frees Centrifuged cells form
infect cells. outside phage a pellet. Free phages
parts from cells. and phage parts remain
in liquid.
Radioactivity
Radioactive (phage protein)
protein
found in liquid
DNA
Centrifuge
Pellet

Figure 13.5-2
Experiment
Batch 2: Radioactive phosphorus (32P) in phage DNA
Labeled phages Agitation frees Centrifuged cells form
infect cells. outside phage a pellet. Free phages
parts from cells. and phage parts remain
in liquid.
Radioactive
DNA
Centrifuge
Radioactivity
Pellet
(phage DNA)
found in pellet

Additional Evidence That DNA Is the Genetic Material
 It was known that DNA is a polymer of nucleotides,

each consisting of a nitrogenous base, a sugar, and
a phosphate group
 In 1950, Erwin Chargaff reported that DNA
composition varies from one species to the next
 This evidence of diversity made DNA a more
credible candidate for the genetic material

Figure 13.6
Sugar- Nitrogenous bases

5 end
phosphate
backbone
Thymine (T)
Adenine (A)
Cytosine (C)
3 end Guanine (G)
DNA
nucleotide

Figure 13.6-1
Phosphate
3 end
Sugar
(deoxyribose)
DNA
nucleotide Nitrogenous base

 Two findings became known as Chargaff’s rules
 The base composition of DNA varies between
species
 In any species the percentages of A and T bases are
equal and the percentages of G and C bases are
equal
 The basis for these rules was not understood until
the discovery of the double helix

Building a Structural Model of DNA: Scientific
Inquiry
 James Watson and Francis Crick were first to
determine the structure of DNA
 Maurice Wilkins and Rosalind Franklin were using a
technique called X-ray crystallography to study
molecular structure
 Franklin produced a picture of the DNA molecule
using this technique

Figure 13.7
(a) Rosalind Franklin (b) Franklin’s X-ray diffraction

photograph of DNA

 Franklin’s X-ray crystallographic images of DNA
enabled Watson to deduce that DNA was helical
 The X-ray images also enabled Watson to deduce
the width of the helix and the spacing of the
nitrogenous bases
 The pattern in the photo suggested that the DNA
molecule was made up of two strands, forming a
double helix

Figure 13.8
5 end
C G
C G Hydrogen bond
3 end
G C
G C T A
3.4 nm
T A
G C G C
C G
A T
1 nm C G
T A
C G
G C
C G A T
A T 3 end
A T
0.34 nm 5 end
T A
(a) Key features of (b) Partial chemical structure (c) Space-filling

DNA structure model

Figure 13.8-1
C G
C G
G C
G C
3.4 nm
T A
G C
C G
A T
1 nm
T A
C G
G C
C G
A T
A T
0.34 nm
T A
(a) Key features of

DNA structure
Figure 13.8-2
5 end
Hydrogen bond
3 end
T A
G C
C G
A T
3 end
5 end
(b) Partial chemical structure
Figure 13.8-3
(c) Space-filling
model
 Watson and Crick built models of a double helix to
conform to the X-ray measurements and the
chemistry of DNA
 Franklin had concluded that there were two outer
sugar-phosphate backbones, with the nitrogenous
bases paired in the molecule’s interior
 Watson built a model in which the backbones were
antiparallel (their subunits run in opposite
directions)

 At first, Watson and Crick thought the bases paired
like with like (A with A, and so on), but such pairings
did not result in a uniform width
 Instead, pairing a purine with a pyrimidine resulted
in a uniform width consistent with the X-ray data

Figure 13.9
Purine + purine: too wide
Pyrimidine + pyrimidine: too narrow
Purine + pyrimidine: width

consistent with X-ray data

 Watson and Crick reasoned that the pairing was
more specific, dictated by the base structures
 They determined that adenine (A) paired only with
thymine (T), and guanine (G) paired only with
cytosine (C)
 The Watson-Crick model explains Chargaff’s rules:
in any organism the amount of A = the amount of T,
and the amount of G = the amount of C

Figure 13.10
Adenine (A) Thymine (T)
Guanine (G) Cytosine (C)

Concept 13.2: Many proteins work together in DNA
replication and repair
 The relationship between structure and function is
manifest in the double helix
 Watson and Crick noted that the specific base
pairing suggested a possible copying mechanism
for genetic material

Figure 13.11-s1
5 3
A T
C G
T A
A T
G C
3 5
(a) Parental
molecule

Figure 13.11-s2
5 3 5 3
A T A T
C G C G
T A T A
A T A T
G C G C
3 5 3 5
(a) Parental (b) Separation of parental

molecule strands into templates

Figure 13.11-s3
5 3 5 3 5 3 5 3
A T A T A T A T
C G C G C G C G
T A T A T A T A
A T A T A T A T
G C G C G C G C
3 5 3 5 3 5 3 5
(a) Parental (b) Separation of parental (c) Formation of new strands

molecule strands into templates complementary to template
strands

The Basic Principle: Base Pairing to a Template
Strand
 Since the two strands of DNA are complementary,
each strand acts as a template for building a new
strand in replication
 In DNA replication, the parent molecule unwinds,
and two new daughter strands are built based on
base-pairing rules

 Watson and Crick’s semiconservative model of
replication predicts that when a double helix
replicates, each daughter molecule will have one
old strand (derived or “conserved” from the parent
molecule) and one newly made strand
 Competing models were the conservative model
(the two parent strands rejoin) and the dispersive
model (each strand is a mix of old and new)

Figure 13.12
First Second
Parent cell replication replication
(a) Conservative model
(b) Semiconservative model
(c) Dispersive model

 Experiments by Matthew Meselson and Franklin
Stahl supported the semiconservative model

Figure 13.13
Experiment
Bacteria Bacteria
cultured in transferred
medium to medium
with15N with14N
(heavy (lighter
isotope) isotope)
Results DNA sample DNA sample Less

centrifuged centrifuged dense
after first after second More
replication replication dense
Conclusion
Predictions: First replication Second replication
Conservative
model
Semiconservative
model
Dispersive
model

Figure 13.13-1
Experiment
Bacteria Bacteria
cultured in transferred
medium to medium
with15N with14N
(heavy (lighter
isotope) isotope)
Results DNA sample DNA sample Less

centrifuged centrifuged dense
after first after second More
replication replication dense

Figure 13.13-2
Conclusion
Predictions: First replication Second replication
Conservative
model
Semiconservative
model
Dispersive
model

DNA Replication: A Closer Look
 The copying of DNA is remarkable in its speed and

accuracy
 More than a dozen enzymes and other proteins
participate in DNA replication
 Much more is known about how this “replication
machine” works in bacteria than in eukaryotes
 Most of the process is similar between prokaryotes
and eukaryotes

Getting Started
 Replication begins at sites called origins of

replication, where the two DNA strands are
separated, opening up a replication “bubble”
 At each end of a bubble is a replication fork, a
Y-shaped region where the parental strands of DNA
are being unwound

Figure 13.14
Primase
Topoisomerase
3
RNA
5
3 primer
5
Replication
3 fork
5
Helicase
Single-strand binding
proteins

 Helicases are enzymes that untwist the double
helix at the replication forks
 Single-strand binding proteins bind to and
stabilize single-stranded DNA
 Topoisomerase relieves the strain caused by tight
twisting ahead of the replication fork by breaking,
swiveling, and rejoining DNA strands

Figure 13.15-1
(a) Origin of replication in an E. coli cell
Parental (template) strand

Origin of
replication Daughter (new)
strand
Replication
Double- fork
stranded
DNA Replication
molecule bubble
Two daughter
DNA molecules
0.5 mm
Figure 13.15-1a
0.5 mm
 Multiple replication bubbles form and eventually
fuse, speeding up the copying of DNA

Figure 13.15-2
(b) Origins of replication in a eukaryotic cell
Origin of Double-stranded
replication DNA molecule
Parental Daughter
(template) strand (new) strand
Bubble Replication fork
Two daughter DNA molecules
0.25 mm
 The enzyme, primase, starts an RNA chain with a
single RNA nucleotide and adds RNA nucleotides
one at a time using the parental DNA as a template
 The primer is short (5–10 nucleotides long)
 The new DNA strand will start from the 3 end of the
RNA primer

 Enzymes called DNA polymerases catalyze the
elongation of new DNA at a replication fork
 Most DNA polymerases require a primer and a DNA
template strand
 The rate of elongation is about 500 nucleotides per
second in bacteria and 50 per second in human
cells

 Each nucleotide that is added to a growing DNA
consists of a sugar attached to a base and to three
phosphate groups
 dATP is used to make DNA and is similar to the
ATP of energy metabolism
 The difference is in the sugars: dATP has
deoxyribose, while ATP has ribose
 As each monomer nucleotide joins the DNA strand,
it loses two phosphate groups as a molecule of
pyrophosphate

Figure 13.16
New strand Template strand

5 3 5 3
Sugar A T A T
Phosphate Base
C G C G
G C DNA G C
poly-
merase
3 A T A
P Pi
C 3 C
Pyro-
phosphate
Nucleotide
5 5
2 Pi
Antiparallel Elongation
 Newly replicated DNA strands must be formed

antiparallel to the template strand
 DNA polymerases add nucleotides only to the free
3 end of a growing strand; therefore, a new DNA
strand can elongate only in the 5 to 3 direction

 Along one template strand of DNA, the DNA
polymerase synthesizes a leading strand
continuously, moving toward the replication fork

Figure 13.17
Leading Overview
Origin of replication Lagging
strand strand
Primer
Leading
Lagging strand Overall strand
directions
of replication
Origin of replication
3
5
RNA primer
5 3 Sliding clamp
3
DNA pol III
5
Parental DNA
3
5
5 Continuous elongation
3 3 in the 5 to 3 direction
5
Figure 13.17-1
Overview
Leading Lagging
strand Origin of replication
strand
Primer
Leading
Lagging strand Overall strand
directions
of replication

Figure 13.17-2
3
5
RNA primer
5 3 Sliding clamp
3
DNA pol III
5
Parental DNA
3
5
5 Continuous elongation
3 3 in the 5 to 3 direction
5
 To elongate the other new strand, the lagging
strand, DNA polymerase must work in
the direction away from the replication fork
 The lagging strand is synthesized as a series of
segments called Okazaki fragments

 After formation of Okazaki fragments, DNA
polymerase I removes the RNA primers and
replaces the nucleotides with DNA
 The remaining gaps are joined together by DNA
ligase

Figure 13.18
Overview
Lagging Origin of replication
Leading strand
Lagging
strand strand
Leading RNA primer

Overall directions strand for fragment 2
of replication 5 Okazaki
3 DNA pol III
3 Primase makes fragment 2
Primer for makes Okazaki
RNA primer. Origin of leading fragment 2.
replication strand
5 3 3
Template 5 3 5
strand 5
5
DNA pol III 3 DNA pol I
3 RNA primer makes Okazaki replaces RNA
for fragment 1 fragment 1. with DNA.
3
5 3 5 3 5
5 DNA ligase forms
bonds between
DNA pol III 5
DNA fragments.
detaches. 3
3
Okazaki
fragment 1 3
5 5
3
5 Overall direction of replication
Figure 13.18-1
Overview
Lagging Origin of replication
strand
Leading Lagging
strand strand
Leading
Overall directions strand
of replication

Figure 13.18-2-s1
3 Primase makes Primer for

RNA primer. Origin of leading
replication strand
5 3
Template 5 3
strand 5

Figure 13.18-2-s2

replication strand
5 3
Template 5 3
strand 5
DNA pol III

3 RNA primer makes Okazaki
for fragment 1 fragment 1.
5 3 5 3
5

Figure 13.18-2-s3

replication strand
5 3
Template 5 3
strand 5
DNA pol III

3 RNA primer makes Okazaki
for fragment 1 fragment 1.
5 3 5 3
5
DNA pol III

detaches.
3
Okazaki
fragment 1
5
3
5

Figure 13.18-3-s1
RNA primer for fragment 2
5 Okazaki
DNA pol III
3 fragment 2
makes Okazaki
fragment 2.
3
5

Figure 13.18-3-s2
5 Okazaki
DNA pol III
3 fragment 2
makes Okazaki
fragment 2.
3
5
5
3 DNA pol I
replaces RNA
with DNA.
3
5

Figure 13.18-3-s3
5 Okazaki
DNA pol III
3 fragment 2
makes Okazaki
fragment 2.
3
5
5
3 DNA pol I
replaces RNA
with DNA.
3
5
DNA ligase forms
bonds between
5 DNA fragments.
3
3
5
Overall direction of replication
The DNA Replication Complex
 The proteins that participate in DNA replication form

a large complex, a “DNA replication machine”
 The DNA replication machine may be stationary
during the replication process
 Recent studies support a model in which two DNA
polymerase molecules “reel in” parental DNA and
“extrude” newly made daughter DNA molecules

Figure 13.20
Leading strand template
DNA pol III

Leading strand
Parental DNA 5
5 3 3
3 3
5 5
Connecting protein
Helicase
DNA pol III
Lagging 3 5
strand Lagging strand
5 3
template
Overall direction of replication

Proofreading and Repairing DNA
 DNA polymerases proofread newly made DNA,

replacing any incorrect nucleotides
 In mismatch repair of DNA, other enzymes correct
errors in base pairing
 A hereditary defect in one such enzyme is
associated with a form of colon cancer
 This defect allows cancer-causing errors to
accumulate in DNA faster than normal

 DNA can be damaged by exposure to harmful
chemical or physical agents such as cigarette
smoke and X-rays
 It can also undergo spontaneous changes
 In nucleotide excision repair, a nuclease cuts out
and replaces damaged stretches of DNA

Figure 13.21-s1
5 3
3 5
Nuclease
5 3
3 5

Figure 13.21-s2
5 3
3 5
Nuclease
5 3
3 5
DNA
polymerase
5 3
3 5

Figure 13.21-s3
5 3
3 5
Nuclease
5 3
3 5
DNA
polymerase
5 3
3 5
DNA
ligase
5 3
3 5
Evolutionary Significance of Altered DNA
Nucleotides
 The error rate after proofreading repair is low but
not zero
 Sequence changes may become permanent and
can be passed on to the next generation
 These changes (mutations) are the source of the
genetic variation upon which natural selection
operates

Replicating the Ends of DNA Molecules
 For linear DNA, the usual replication machinery

cannot complete the 5 ends of daughter strands
 Repeated rounds of replication produce shorter
DNA molecules with uneven ends
 Eukaryotic chromosomal DNA molecules have
special nucleotide sequences at their ends called
telomeres

Figure 13.22
1 mm

 Telomeres typically consist of multiple repetitions of
one short nucleotide sequence
 Telomeres do not prevent the shortening of DNA
molecules, but they do postpone it
 It has been proposed that the shortening of
telomeres is connected to aging

 If chromosomes of germ cells became shorter in
every cell cycle, essential genes would eventually
be missing from the gametes they produce
 An enzyme called telomerase catalyzes the
lengthening of telomeres in germ cells

 Telomerase is not active in most human somatic
cells
 However, it does show inappropriate activity in
some cancer cells
 Telomerase is currently under study as a target for
cancer therapies

Concept 13.3: A chromosome consists of a DNA
molecule packed together with proteins
 The bacterial chromosome is a double-stranded,
circular DNA molecule associated with a small
amount of protein
 Eukaryotic chromosomes have linear DNA
molecules associated with a large amount of protein
 In a bacterium, the DNA is “supercoiled” and found
in a region of the cell called the nucleoid

 Chromatin, a complex of DNA and protein, is found
in the nucleus of eukaryotic cells
 Chromosomes fit into the nucleus through an
elaborate, multilevel system of packing
 Chromatin undergoes striking changes in the
degree of packing during the course of the cell cycle

 Proteins called histones are responsible for the first
level of DNA packing in chromatin
 Four types of histones are most common in
chromatin: H2A, H2B, H3, and H4
 A nucleosome consists of DNA wound twice
around a protein core of eight histones, two of each
of the main histone types

Figure 13.23
Chromatid
(700 nm)
Nucleosome
DNA (10 nm in diameter)
30-nm
double helix fiber
(2 nm in diameter)
Looped
Scaffold
domain
H1 300-nm
Histone tail
Histones fiber
Replicated chromosome
(1,400 nm)

Figure 13.23-1
Nucleosome
(10 nm in diameter)
DNA
double helix
(2 nm in diameter)
H1
Histone tail
Histones

Figure 13.23-1a
DNA
double helix
(2 nm in diameter)

Figure 13.23-1b
Nucleosome
(10 nm in diameter)

Figure 13.23-2
Chromatid
(700 nm)
30-nm
fiber
Looped Scaffold
domain
300-nm
fiber
Replicated chromosome
(1,400 nm)
Figure 13.23-2a
30-nm fiber

Figure 13.23-2b
Looped Scaffold
domain

Figure 13.23-2c
Chromatid
(700 nm)

 At interphase, most of the chromatin is compacted
into a 30-nm fiber, which is folded further in some
areas by looping
 Even during interphase, centromeres and some
other parts of chromosomes are highly condensed,
similar to metaphase chromosomes
 This condensed chromatin is called
heterochromatin; the more dispersed, less
compacted chromatin is called euchromatin

 Dense packing of the heterochromatin makes it
largely inaccessible to the machinery responsible
for transcribing genetic information
 Chromosomes are dynamic in structure; a
condensed region may be loosened or modified as
needed for various cell processes
 For example, histones can undergo chemical
modifications that result in changes in chromatin
organization

Concept 13.4: Understanding DNA structure and
replication makes genetic engineering possible
 Complementary base pairing of DNA is the basis for
nucleic acid hybridization, the base pairing of one
strand of a nucleic acid to another, complementary
sequence
 Nucleic acid hybridization forms the foundation of
virtually every technique used in genetic
engineering, the direct manipulation of genes for
practical purposes

DNA Cloning: Making Multiple Copies of a Gene or
Other DNA Segment
 To work directly with specific genes, scientists
prepare well-defined segments of DNA in identical
copies, a process called DNA cloning
 Most methods for cloning pieces of DNA in the
laboratory share general features

 Many bacteria contain plasmids, small circular
DNA molecules that replicate separately from the
bacterial chromosome
 To clone pieces of DNA, researchers first obtain a
plasmid and insert DNA from another source
(“foreign DNA”) into it
 The resulting plasmid is called recombinant DNA

Figure 13.24
Bacterium Gene inserted into plasmid Cell containing gene
(a cloning vector) of interest
Bacterial Plasmid
chromosome DNA of chromosome
(“foreign” DNA)
Gene of interest
Recombinant
DNA (plasmid) Plasmid put into
bacterial cell
Recombinant
bacterium
Host cell grown in culture to form a clone of
cells containing the “cloned” gene of interest
Gene of
interest
Protein expressed
from gene of interest
Copies of gene Protein harvested
Basic
research
Gene for pest resistance and various Human growth hormone
inserted into plants applications treats stunted growth
Gene used to alter bacteria Protein dissolves blood clots

for cleaning up toxic waste in heart attack therapy
Figure 13.24-1
Bacterium Gene inserted into plasmid Cell containing

(a cloning vector) gene of interest
Bacterial Plasmid
chromosome DNA of
chromosome
(“foreign” DNA)
Recombinant Gene of interest
DNA (plasmid) Plasmid put into
bacterial cell
Recombinant
bacterium
Host cell grown in culture to
form a clone of cells containing
the “cloned” gene of interest
Gene of
interest
Protein expressed

Figure 13.24-2
Gene of
interest
Protein expressed
Copies of gene Protein harvested
Basic
research
and various
applications
Gene for pest resistance Human growth hormone

inserted into plants treats stunted growth
Gene used to alter bacteria Protein dissolves blood clots

for cleaning up toxic waste in heart attack therapy
 The production of multiple copies of a single gene is
called gene cloning
 The plasmid that carries the cloned DNA is called a
cloning vector
 Gene cloning is used to make many copies of a
gene and to produce a protein product
 The ability to amplify many copies of a gene is
crucial for applications involving a single gene

Using Restriction Enzymes to Make Recombinant
DNA
 Bacterial restriction enzymes cut DNA molecules
at specific DNA sequences called restriction sites
 A restriction enzyme usually makes many cuts,
yielding restriction fragments

Animation: Restriction Enzymes

Figure 13.25
Bacterial
plasmid
Restriction site
5 3
DNA GA ATTC
C.T TAAG
3 5
Restriction enzyme cuts
the sugar-phosphate
backbones at each arrow.
3 5
5 G
3
5 3
3 Sticky 5
end
5
3
5
DNA fragment from another 3
source is added. Base pairing Fragment from different
of sticky ends produces
DNA molecule cut by the
various combinations.
same restriction enzyme
5 35 35 3
G AAT T C G AATT C
C T TAA G C TTAA G
3 53 53 5
One possible combination
DNA ligase
seals the strands.
5 3
3 Recombinant DNA molecule 5
Recombinant
plasmid
Figure 13.25-1
Bacterial
plasmid
Restriction site
5 3
DNA G A AT T C
C T T AAG
3 5
Restriction enzyme cuts
the sugar-phosphate
backbones at each arrow.
3 5
5 G
3
5 3
3 Sticky 5
end
Figure 13.25-2
3 5
5 3
5 3
3 Sticky 5
end
5
3
5
DNA fragment from another 3
source is added. Base pairing
of sticky ends produces Fragment from different
various combinations. DNA molecule cut by the
same restriction enzyme
5 3 5 3 5 3
G AAT T C G AAT T C
C T TA A G C T TA A G
3 53 53 5

Figure 13.25-3
5 3 5 3 5 3
G AAT T C G AAT T C
C T TAA G C T TAA G
3 53 53 5
DNA ligase
seals the strands.
5 3
3 Recombinant DNA molecule 5
Recombinant
plasmid

 The most useful restriction enzymes cleave the
DNA in a staggered manner to produce sticky
ends
 Sticky ends can bond with complementary sticky
ends of other fragments
 DNA ligase can close the sugar-phosphate
backbones of DNA strands

 To see the fragments produced by cutting DNA
molecules with restriction enzymes, researchers
use gel electrophoresis
 This technique separates a mixture of nucleic acid
fragments based on length

Figure 13.26
Mixture of Power
DNA mol- source
ecules of Cathode Anode
different
lengths
Wells
Gel
(a) Negatively charged DNA molecules will move

toward the positive electrode.
Restriction fragments
of known lengths
(b) Shorter molecules are slowed down less than

longer ones, so they move faster through the gel.
Figure 13.26-1
Mixture of Power
DNA mol- source
ecules of Cathode Anode
different
lengths
Wells
Gel
(a) Negatively charged DNA molecules will move

toward the positive electrode.

Figure 13.26-2
Restriction fragments
of known lengths
(b) Shorter molecules are slowed down less than

longer ones, so they move faster through the gel.

Amplifying DNA in Vitro: The Polymerase Chain
Reaction (PCR) and Its Use in Cloning
 The polymerase chain reaction (PCR) can
produce many copies of a specific target segment of
DNA
 A three-step cycle brings about a chain reaction that
produces an exponentially growing population of
identical DNA molecules
 The key to PCR is an unusual, heat-stable DNA
polymerase called Taq polymerase.

Figure 13.27
Technique 5 3
Target sequence
Genomic DNA 3 5
Denaturation 5 3
3 5
Annealing
Cycle 1
yields 2 molecules Primers
Extension
New
nucleotides
Cycle 2
yields 4 molecules
Cycle 3
2 of the 8 molecules
(in white boxes)
match target sequence
and are the right length
Figure 13.27-1
Technique
5 3
Target sequence
Genomic DNA 3 5

Figure 13.27-2-s1
3 5
Cycle 1
yields 2
molecules

Figure 13.27-2-s2
3 5
Annealing
Cycle 1
yields 2
molecules Primers

Figure 13.27-2-s3
3 5
Annealing
Cycle 1
yields 2
molecules Primers
Extension
New
nucleotides

Figure 13.27-3
Cycle 2
yields 4 molecules
Cycle 3
2 of the 8 molecules
(in white boxes)
match target sequence
and are the right length
Results After 30 more cycles, over 1 billion (109) molecules

match the target sequence.

 PCR amplification alone cannot substitute for gene
cloning in cells
 Instead, PCR is used to provide the specific DNA
fragment to be cloned
 PCR primers are synthesized to include a restriction
site that matches the site in the cloning vector
 The fragment and vector are cut and ligated
together

Figure 13.28
Cloning DNA fragment obtained

vector by PCR (cut by same
(bacterial restriction enzyme used
plasmid) on cloning vector)
Mix and ligate
Recombinant
DNA plasmid

DNA Sequencing
 Once a gene is cloned, complementary base pairing

can be exploited to determine the gene’s complete
nucleotide sequence
 This process is called DNA sequencing

 “Next-generation” sequencing techniques,
developed in the last 15 years, are rapid and
inexpensive
 They sequence by synthesizing the complementary
strand of a single, immobilized template strand
 A chemical technique enables electronic monitors to
identify which nucleotide is being added at each
step

Figure 13.29
(a) Next-generation sequencing machines
4-mer A
T
3-mer G TTCT GCG AA
C
2-mer
1-mer
(b) A “flow-gram” from a next-generation

sequencing machine
Figure 13.29-1
(a) Next-generation sequencing machines

Figure 13.29-2
4-mer A
T
3-mer G TTC TGCG AA
C
2-mer
1-mer
(b) A “flow-gram” from a next-generation sequencing

machine

 Next-generation methods are being complemented
or replaced by third-generation methods
 These newer techniques are faster and less
expensive
 Several groups are working on “nanopore”
methods, which involve moving a single DNA strand
through a tiny pore in a membrane
 Nucleotides are identified by slight differences in the
amount of time that they interrupt an electrical
current across the pore

Figure 13.30

Editing Genes and Genomes
 Over the past five years, biologists have developed

a powerful new technique called the CRISPR-Cas9
system
 Cas9 is a nuclease that cuts double-stranded DNA
molecules as directed by a guide RNA that is
complementary to the target gene
 Researchers have used this system to “knock out”
(disable) a given gene in order to determine its
function

 Researchers have also modified the CRISPR-Cas9
system to repair a gene that has a mutation
 In 2014 a group of researchers reported using this
system to successfully correct a mutated gene in
mice
 CRISPR technology is sparking widespread
excitement among researchers and physicians

Figure 13.31
Cas9 active sites NUCLEUS

Guide RNA
complementary
sequence
Cas9 protein Guide RNA engineered to 5 3
“guide” the Cas9 protein 5
3 5
to a target gene
5 Part of the
3 target gene
Complementary
sequence that can Resulting cut
Active sites that bind to a target gene in target gene
can cut DNA
Cas9-guide RNA complex
Normal
(functional)
gene for use as
a template by
repair enzymes
(a) Scientists can disable (b) If the target gene has a
CYTOPLASM (“knock out”) the target gene mutation, it can be repaired.
to study its normal function.
NUCLEUS
Random nucleotides Normal nucleotides

Figure 13.31-1
Cas9 protein Guide RNA engineered to

“guide” the Cas9 protein
to a target gene
5
3
Complementary
sequence that can
Active sites that bind to a target gene
can cut DNA
Cas9-guide RNA complex

Figure 13.31-2
CYTOPLASM
Cas9 active sites NUCLEUS

Guide RNA
complementary
sequence
5 3
5
3 5
Part of the
target gene
Resulting cut
in target gene

Figure 13.31-3
Normal
(functional)
gene for use as
a template by
repair enzymes
(a) Scientists can disable (b) If the target gene has a

(“knock out”) the target gene mutation, it can be repaired.
to study its normal function.
Random nucleotides Normal nucleotides

Figure 13.15
(a) Origin of replication in an E. coli cell (b) Origins of replication in a eukaryotic cell
Parental (template) strand Origin of Double-stranded

Origin of replication DNA molecule
replication Daughter (new)
strand Parental Daughter
Replication (template) strand (new) strand
Double- fork
stranded
DNA Replication
molecule bubble Bubble Replication fork
Two daughter
DNA molecules
Two daughter DNA molecules
0.25 mm
0.5 mm

Figure 13.19
Overview
Leading strand Lagging
Leading strand strand
template
Single-strand 3
Leading
binding proteins Lagging strand strand
Overall directions 5
of replication
Leading strand
Helicase
5 DNA pol III
3 Primer
3 5 3 Primase Lagging strand
Parental DNA DNA pol III
5 DNA pol I
Lagging strand 3 DNA ligase
template 5
3
5

Figure 13.19-1
Overview
Leading strand Lagging
strand
Leading
Lagging strand strand
Overall directions
of replication

Figure 13.19-2
Leading strand
template
Single-strand
binding proteins
Helicase Leading strand
5 DNA pol III

3 Primer
3 5 3 Primase
Parental DNA
Lagging strand
template

Figure 13.19-3
Lagging strand
DNA pol III
5 DNA pol I DNA ligase
3
5 3
Lagging strand template 5

Figure 13.UN01-1

Figure 13.UN03
DNA pol III synthesizes

leading strand continuously
3
5
Parental
DNA DNA pol III starts DNA
synthesis at 3 end of primer, Origin of
5 continues in 5 → 3 direction replication
3
Lagging strand synthesized
in short Okazaki fragments,
Helicase
later joined by DNA ligase
3
Primase synthesizes 5
a short RNA primer
DNA pol I replaces the RNA
primer with DNA nucleotides

Figure 13.UN04
5 3 5 3
G A AT TC
C T T AA G
3 5 3 5
Sticky end

Figure 13.UN05

Figure 13.UN06

Molecular Basic of Inheritance

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CAMPBELL BIOLOGY IN FOCUS

URRY • CAIN • WASSERMAN • MINORSKY • REECE

© 2016 Pearson Education, Inc. SECOND EDITION

 In 1953, James Watson and Francis Crick

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 Early in the 20th century, the identification of the

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 The discovery of the genetic role of DNA began with

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Mouse dies Mouse healthy Mouse healthy Mouse dies

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 More evidence for DNA as the genetic material

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 It was known that DNA is a polymer of nucleotides,

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Sugar- Nitrogenous bases

3 end Guanine (G)

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(a) Rosalind Franklin (b) Franklin’s X-ray diffraction

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(a) Key features of (b) Partial chemical structure (c) Space-filling

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(a) Key features of

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Purine + purine: too wide

Pyrimidine + pyrimidine: too narrow

Purine + pyrimidine: width

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Adenine (A) Thymine (T)

Guanine (G) Cytosine (C)

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(a) Parental (b) Separation of parental

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(a) Parental (b) Separation of parental (c) Formation of new strands

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(b) Semiconservative model

(c) Dispersive model

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Results DNA sample DNA sample Less

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Results DNA sample DNA sample Less

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 The copying of DNA is remarkable in its speed and

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